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Immunogenicity of xenopeptide hormone therapies
peptides 27 (2006) 1902–1910
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Review
Immunogenicity of xenopeptide hormone therapies Catherine A. Schnabel a,*, S. Edwin Fineberg b, Dennis D. Kim a a b
Amylin Pharmaceuticals, Inc., 9360 Towne Centre Dr., San Diego, CA 92121, USA Indiana University School of Medicine, Indianapolis, IN 46202, USA
article info
abstract
Article history:
Peptides are a growing class of agents whose therapeutic use originated with non-human
Received 2 November 2005
treatments such as animal insulins. Xenopeptides continue to be explored for biothera-
Received in revised form
peutic development using genetic engineering, and through the rich resource of animal and
25 January 2006
plant polypeptides. One of the major concerns of therapeutic administration of xenopep-
Accepted 26 January 2006
tides is the potential for untoward immune responses that may lead to loss of drug efficacy
Published on line 3 March 2006
or adverse events in recipients. An increased risk of immunogenicity is perceived with xenopeptides, however, human-derived therapies also induce antibody formation that in
Keywords:
some cases has been associated with severe clinical sequelae. In this review, antibody
Immunogenicity
responses to xenopeptides are highlighted looking at current hormone therapies used to
Xenopeptide
treat endocrine disorders. Similar to clinical experiences with peptide-based agents in
Hormone therapies
general, antibody responses against xenopeptide hormone therapies in majority of cases have been benign in nature with minimal clinical impact. # 2006 Elsevier Inc. All rights reserved.
Contents 1. 2. 3. 4.
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1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune responses to protein therapeutics . . . Variables of antibody induction and detection . Xenoprotein therapies . . . . . . . . . . . . . . . . . . . . 4.1. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Calcitonin . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Exenatide . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction
Protein therapeutics have revolutionized the treatment of a wide variety of diseases [36]. Because of their specificity,
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peptide-based agents comprise an important component of the therapeutic arsenal, and have the potential to provide efficacy with minimal adverse effects. A significant barrier to the use of protein therapeutics is the development of
* Corresponding author. Tel.: +1 858 458 8464; fax: +1 858 824 7628. E-mail address: [email protected] (C.A. Schnabel). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.01.019
peptides 27 (2006) 1902–1910
unwanted anti-drug immune reactions. Nearly all protein drugs have the potential for antibody induction, although the incidence and associated clinical consequences vary widely among agents [19,45,58,90,91]. In the case of therapies intended for chronic therapy, surveillance for adverse immunological effects are particularly important since they may contribute to complications associated with long-term use. Concern over untoward immunological effects of protein therapeutics was initially brought to the forefront when the first proteins purified from animal tissue extracts became available for therapeutic use [6]. Since non-native molecules were perceived to be more antigenic than their human counterparts, the extent of sequence divergence from human homologs was thought to fully explain their immunogenic properties. However, antibody formation was subsequently shown in recipients treated with protein therapies isolated from human tissues or sera [53,85], including recipients who were not innately deficient for a given protein [93]. Over time, it has become increasingly apparent that factors influencing drug-related immunogenicity are not fully understood, or completely predictable.
2.
Immune responses to protein therapeutics
Immunogenicity of therapeutic proteins has largely been defined by the presence of circulating antibodies in clinical samples. Various assay formats are used to assess immunogenicity. First-line assays used to measure antibody levels include enzyme-linked immunosorbant assays (ELISAs), radioimmunoassays (RIAs) and more sophisticated methods such as surface plasmon resonance (SPR). Additional qualitative information on antibody specificity and immune responses is attainable through lower-throughput methodologies including immunoblotting, ex vivo T-cell activation assays and animal studies in genetically engineered mouse models. Further characterization of potential neutralizing activity is typically achieved through cell-based bioassays. Distinct advantages and disadvantages are associated with the specific assay type, each requiring appropriate design, customization and validation [19,20,59,103]. Immune responses to protein drugs can essentially be viewed as an active immunization or a failure of immune tolerance, depending on whether the immune system encounters a unique presentation of an antigen. B-cell maturation and high affinity antibody production ensue through MHC-mediated antigen presentation, T-cell activation and cytokine stimulation. Therapies of non-human origin, such as those from animals, plants or microbes, elicit the formation of foreign-antigen-specific antibodies. Generally, antibodies mounted against such antigens develop as an IgG isotype upon antibody maturation, and can be produced and persist even after a single dose [91]. In comparison, antibody responses against human proteins result from breaking tolerance mechanisms that exist for self-antigens. Immune tolerance may result from active suppression of Bcell maturation by regulatory T-cells or through the destruction or sequestration of antigen specific T-cells [73]. Disruption of tolerance has been related to alterations in the physical properties of the drug, the spread of immunological sequelae
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during intercurrent illness, or the use of immunity-modifying drugs, however, precise underlying mechanisms leading to the breaking of tolerance are not completely understood [8,12,92]. These antibodies may be binding, with or without neutralizing activity, and appear after prolonged treatment [90]. In some cases, anti-drug antibodies are observed in a minority of patients and are undetectable after treatment cessation [99]. The impact of antibody formation against protein therapeutics ranges from having no clinical effect to lifethreatening conditions [58]. Antibody incidences also range widely, with less then 1% observed for some products [43] to >70% for others [42]. Among various protein therapeutics associated with immunogenicity, reports of deleterious effects include those where antibodies neutralize an endogenous counterpart with essential, non-redundant functions. The most notable and severe occurrence of this was an outbreak of antibody-mediated pure red cell aplasia (PRCA) in patients treated with recombinant human erythropoietin (rHuEpo) [4,15]. Between 1999 and 2001, hundreds of cases of erythropoietin-associated red cell aplasia were identified, with a majority of cases involving patients with chronic kidney disease receiving subcutaneous injections of erythropoietin alpha [89]. Anti-rHuEpo antibodies, which neutralized not only the recombinant protein, but also native erythropoietin, led to the absence of red cell precursors in the bone marrow of patients, rendering them transfusion-dependent [15]. A change in formulation in 1998 was thought to be the reason for the induction of PRCA. Reduced incidences of PRCA reported over the last 3 years have been attributed to a change in the route of administration and revised storage and handling protocols [92]. In another example, a small proportion of healthy platelet donors developed thrombocytopenia when treated with Megakaryocyte Growth and Differentiation Factor (PEG-MGDF) [62]. Incidences of drug-induced illness resembling autoimmune disorders have also been described in association with protein therapies [74]. In particular, druginduced lupus has been reported as an adverse effect of antiTumor Necrosis Factor-a agents as well as numerous other non-protein drugs [5,87]. Clinical and laboratory features of drug-induced lupus are similar to systemic lupus erythematosus except that the syndrome typically reverts after druguse is stopped [5,74]. Taken together, these more serious instances, although relatively less common, have raised increasing concern over the potential harmful effects of antibody formation in response to protein therapeutics. Additional complications may arise from IgE-mediated immune responses. Drug-induced allergies vary from resolvable local responses such as skin reactions to systemic reactions such as anaphylaxis that are potentially fatal. Incidences of severe immediate hypersensitivity reactions are relatively rare and more commonly associated with xenopeptides or drug re-exposure [40]. In addition to adverse clinical implications, antibody production may attenuate drug efficacy and alter pharmacokinetic and pharmacodynamic properties. Anti-drug antibodies have been implicated in clinical resistance [42,68,104,101], to the extent that further treatment becomes ineffective even at high doses of the therapeutic agent [7]. Additionally, antigen–antibody complexes are associated with alterations
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peptides 27 (2006) 1902–1910
in the clearance rate and plasma half-life of drugs, which pose additional safety and efficacy issues [58].
3. Variables of antibody induction and detection Many factors have been implicated in the immunogenic potential of therapeutic proteins (Table 1), which have been reviewed extensively elsewhere [19,58,88,90,91]. Aside from factors that affect the intrinsic response, aspects related to the measurement and characterization of antibody production are also important. Currently, standardization of detection methodologies as well as reporting of antibody levels is lacking [69]. Therefore, comparison of results across laboratories and between manufacturers, and the interpretation of relative immunogenicity, remains a challenge. In sum, drug-induced antibody induction and its associated clinical implications depend on a matrix of factors. A variety of techniques have been developed to address the issue of immunogenicity associated with protein-based therapeutics [19,66]. Aside from humanizing protein sequences in chimeric molecules, efforts to reduce immunogenicity through rational protein engineering have included removal of antibody or MHC-binding epitopes, or sterically inhibiting antibody binding by derivatizing with polyethylene glycol [16,19]. Other patient-based strategies have included prevention of an immune response in recipients by cotreatment with immunosuppressive therapy or by inducing tolerance to limit immune reaction [53]. However, since multiple mechanisms that may or may not be addressable potentially contribute to a drug-induced immune response, long-term monitoring of immunogenicity during clinical trials and during the post-marketing period is essential.
4.
Xenoprotein therapies
The origins of peptide-based therapeutic proteins are as diverse as the many indications for which they have been utilized. Non-human protein sequences from microbes and plants have been used to engineer a variety of hybrid molecules for therapeutic use. The development of chimeric
and humanized monoclonal antibodies has provided unique methods of disease treatment, particularly for antineoplastic and immunological indications. In contrast, protein therapies derived from non-human sources that are essentially homologs or mimetics replicate a native human counterpart in function and regulation, and have been developed as pharmacological replacements. Therapies from non-human sources, or xenoproteins, continue to be explored to exploit novel functions or to provide superior clinical features. A current example of this is desmoteplase, a plasminogen activator (PA) derived from the saliva of the blood-feeding vampire bat (Desmodus rotundus) [3,37]. Desmoteplase exhibits neurotoxicological properties superior to human tissue-PA and is currently being evaluated in clinical trials for the treatment of acute ischemic stroke [41]. The intriguing feature of such agents is that they have been optimized by natural selection, rather than through rational drug design. Antibody responses against hormone therapies have been described [6,35,60,68,86,93,104]. In this review, representative therapies are highlighted to illustrate the implications of antibody formation against xenopeptide hormones currently used to treat chronic diseases.
4.1.
Insulin
Insulin replacement therapy is the mainstay pharmacological treatment for patients with type 1 diabetes and with advanced type 2 diabetes [27]. Immunological complications associated with insulin therapy have been evident since its introduction as a treatment for diabetes in the 1920s. The first insulins were either single species or mixtures of bovine and porcine insulins that were isolated from pancreata. Insulin, which is a 51 amino acid peptide hormone, consists of a 21 amino acid alpha-chain linked by disulfide bonds to a 30-residue betachain. Porcine and human insulin differ by a single amino acid in the beta-chain whereas bovine insulin differs from porcine and human insulin by an additional two amino acids in the alpha-chain [98]. The in vitro use of radioactive insulin in the 1950s showed that virtually 100% of patients treated with animal insulins produced high levels of circulating antiinsulin antibodies [6]. High titer IgG insulin antibodies were shown to bind and neutralize insulin, leading to immunological insulin-resistance by interfering with receptor binding
Table 1 – Factors contributing to increased immunogenic potential of protein therapies Factor
Variable
Increased immunogenic potenial
Intrinsic drug-related factors
Primary sequence Post-translational modification
Non-self antigens Non-native glycosylation
Extrinsic drug-related factors
Manufacturing Purity Formulation Degradation
Presence of contaminants Aggregation Physical, oxidation, deamidation, isomerization
Administration Regimen Method
Longer duration or intermittent treatment Subcutaneous or intradermal > intravenous or oral
Genetic Immune
Specific HLA genotype Impaired or suppressed immunity
Patient-related factors
peptides 27 (2006) 1902–1910
[6,101]. Animal insulins were also commonly associated with hypersensitivity reactions of lipoatrophy and urticaria, and IgE-mediated responses were observed in up to 30% of recipients, however, severe systemic immunological reactions were observed in less than 0.1% of patients [22,101]. In addition to amino acid composition, purity was a major problem with early preparations of animal insulins, which were contaminated with several other islet cell peptides such as proinsulin, C-peptide and glucagon [17]. For instance, in a large study of insulin-dependent patients with diabetes, drug-induced autoimmunity was attributed to antibody formation against hormonal contaminants including glucagon, pancreatic polypeptide and vasoactive intestinal peptide in patients treated with conventional versus monocomponent insulin [11]. The prevalence and titers of anti-insulin antibodies have decreased in the last three decades, largely as a consequence of improvements in the purification of insulin preparations and the development of monocomponent insulins [31,101,104]. To a lesser extent, the availability of recombinant human forms of insulin also contributed to decreased immunogenicity. However, numerous studies have confirmed that recombinant human insulin is also immunogenic [31,32,34]. Insulin antibodies were initially detected 1–2 months after starting treatment and were observable at 2 years in some long-term studies [31,32,93,102]. Although animal insulins used for therapy vary by up to three amino acids, studies indicate that the insulin antibodies produced are not specific for variant residues, but react with determinants shared by the human protein [80,49,65]. A study of insulin naı¨ve patients with type 1 or type 2 diabetes treated exclusively with recombinant human insulin reported approximately 40% of patients were antibody-positive at 1 year [32]. In patients with type 1 diabetes, studies have shown a relatively higher incidence of insulin antibodies in children compared with adults after treatment with both human and animal insulins [93,65]. In one study, antibody prevalence rates of approximately 70% and 90% were reported at 1 and 2 years, respectively, in children treated with human semisynthetic insulin [65]. Despite decreased immunogenicity with human insulin, antibody-positive patients continue to be at risk for altered insulin pharmacokinetics and immunological insulin resistance [52]. Clinical consequences of antibody-induced phenomena may include increases in daily insulin requirements, loss of postprandial glucose control and delayed hypoglycemia, but in large scale trials, the presence of anti-insulin antibodies have not been shown to negatively impact long-term glycemic control directly [101]. Both IgG and IgE insulin antibodies have been detected with human insulin treatment, however in low titers and with decreasing incidences in patients treated exclusively with human insulin [102]. Specifically, the high incidence of systemic allergic reactions and immunologicallymediated insulin resistance observed with non-purified bovine or porcine preparations were reported to occur in less than 0.1% of patients treated de novo with human insulin [90,93]. Many studies have compared human insulin with purified porcine insulin [31,32,47,48,65,83]. Comparative studies reveal a general consensus of their similar potency [83,2,46]. In particular, an integrative analysis of results from 45 randomized controlled clinical trials by Richter and Neises com-
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Fig. 1 – Representative profiles of antibody incidences in response to long-term treatment with human (N = 100) or porcine (N = 121) subcutaneous insulin in patients with type 1 or type 2 diabetes, intranasal salmon calcitonin in patients with postmenopausal osteoporosis (N = 44) or exenatide in patients with type 2 diabetes (N = 357). Adapted from Refs. [1,32,82].
pared the efficacy and adverse event profile between human and animal insulins [83]. Most studies did not detect a significant difference in metabolic control, as defined by measures of glycosylated hemoglobin (HbA1c), fasting plasma glucose and insulin use, notwithstanding heterogeneous study designs and demographics [83]. With regard to immunogenicity, studies have demonstrated that insulin antibody levels are lower in patients using human insulin, with approximately 40–50% incidence compared to 60% with purified porcine insulin [31,32,34] (Fig. 1). Interpretations regarding the clinical significance of the decreased immunogenic potential of human insulin have been varied. An early study in children newly diagnosed with type 1 diabetes indicated adverse effects on residual beta-cell function in the presence of low levels of insulin antibodies [64]. In a larger, controlled study, however, differences in beta-cell function could not be correlated to insulin antibody levels comparing newly diagnosed children treated with either human monocomponent insulin or porcine monocomponent insulin [65]. Taken together, comparative studies indicate that the prevalence and titers of antibodies detected in patients treated with human insulin are lower than with porcine insulin, however the decreased immunogenic potential of human insulins generally has minimal effects on metabolic control. In most Westernized countries, the use of animal insulins has been largely replaced with recombinant human insulins.
4.2.
Calcitonin
Effects of calcitonin to suppress osteoclast-mediated bone resorption have led to its use in treating a variety of bone disorders such as Paget’s disease, postmenopausal
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peptides 27 (2006) 1902–1910
osteoporosis and osteogenesis imperfecta [79]. In patients, long-term treatment with calcitonin has been shown to increase bone mineral density, reduce vertebral and nonvertebral fractures and serve as an analgesic [51]. Calcitonins from several species including human, porcine, salmon and eel have demonstrated clinical value in treating disorders of bone and calcium metabolism [51]. Sequence comparisons among the various therapeutic calcitonins reveal that these 32 amino acid peptides share eight conserved residues (positions 1, 4–7, 9, 28 and 32), with 6 of these clustering at the N-terminus of the molecule [78,71]. Specific divergent residues comprising salmon and eel peptides allow more conformational flexibility, which is a major determinant of biological potency [107]. The activities of salmon and eel calcitonins are approximately 40% more potent than the mammalian calcitonins in lowering plasma calcium and reducing markers of bone turnover [54]. The fish calcitonins, particularly salmon, have achieved more widespread therapeutic use worldwide than human calcitonin due to their increased in vivo potencies and pharmacokinetic properties [50]. Porcine and salmon calcitonins (pCT, sCT) were the first hormones commercially available to treat metabolic diseases characterized by excessive bone resorption [10]. Antibody production during calcitonin therapy was suspected when recipients exhibited a pattern of initial responsiveness with subsequent calcitonin resistance during chronic subcutaneous treatment [95,105]. Studies in patients with Paget’s disease treated with either pCT and/or sCT reported similar prevalences of antibody formation in recipients, ranging from approximately 30% to 40% with sCT and from 60% to 70% with pCT [26,42]. Anti-CT antibodies were detectable approximately 4–12 months after initation of therapy. Upon cessation of therapy, antibodies were undetectable in some patients after 6 months, while in others antibodies persisted for up to 24 months [26]. The most reported outcome of antibody development during heterologous CT therapy is acquired clinical resistance, with antibodies of the IgG class as the major immunological reactants [95,97]. Cases of clinical resistance to sCT are generally correlated to the presence of high-titer anti-sCT antibodies [96]. The clinical significance of CT immunogenicity remains controversial, however, since secondary resistance in some instances is not associated with antibody production [96]. In one study of 15 patients with Paget’s disease treated with pCT or sCT for periods ranging from 4 to 24 months, 6 patients developed secondary resistance related to the presence of neutralizing anti-sCT or anti-pCT antibodies [42]. Another study of 21 patients with Paget’s disease treated with sCT from 1.5 to 19 months reported antibodies in 50% of patients [95]. Whereas three patients developed high titer (>1/ 500) anti-sCT antibodies, only one patient exhibited clinical resistance that could not be explained by dose or treatment duration [95]. Interestingly, cases of secondary resistance to sCT in patients with Paget’s disease have been reported in the absence of circulating anti-sCT antibodies [67], indicating that the occurrence of antibodies does not necessarily correlate with loss of sCT activity. Aside from studies with parenterally administered therapy, the occurrence of antibodies has also been described in
patients treated with nasal sCT [38,61,82]. Reported incidences in osteoporetic patients treated with nasal sCT were comparable to values observed with sCT injection [70]. Similarly, the presence of neutralizing antibodies against sCT administered intranasally in some patients with postmenopausal osteoporosis did not strictly correlate with a loss of therapeutic efficacy [81,82]. Another study of nine patients with Paget’s disease treated for 1 year with nasal sCT showed that all five patients previously treated with injectable sCT developed clinical resistance associated with neutralizing antibody formation within 4 months, indicating that prior exposure may potentiate neutralizing antibody formation [61]. Patients showing clinical resistance to pCT or sCT have been treated effectively with human CT (hCT) [84,63]. Antibody development to hCT is reportedly rare [38,39,97]. Of those reported, antibodies against hCT have been detected in two patients treated for Paget’s disease (one subcutaneous sCT, one intranasal sCT) and in one other treated with subcutaneous sCT for osteoporosis [25,38,39]. sCT therapy currently remains a treatment option for Paget’s disease, osteoporosis and other hypercalcemic conditions. Antibody production against sCT during long-term treatment is undoubtedly common. In general, the presence of low-titer antibodies has no clinical consequences and secondary resistance has been described in the absence of antibodies. Therefore, treatment decisions surrounding sCT should not be dictated solely by the presence of antibodies, but require clinical confirmation evidenced by a parallel loss of therapeutic efficacy.
4.3.
Exenatide
A novel agent recently approved for the treatment of type 2 diabetes, exenatide, is a peptide originally isolated from the salivary secretions of the Gila monster (Heloderma suspectum) [30,72]. Exenatide is a 39 amino acid peptide that shares several glucoregulatory properties with human GLP-1, an incretin hormone released by intestinal cells in response to a meal that augments glucose-dependent insulin secretion [72,75,76]. The actions of exenatide, which target several abnormalities of type 2 diabetes, include the enhancement of glucose-dependent insulin secretion, suppression of inappropriately elevated glucagon secretion, slowing of gastric emptying (which may be paradoxically accelerated in the diabetic state) and reduction in food intake [28,56,57,9,100,29,24]. Improvements in glycemic control with exenatide have been demonstrated as reductions in fasting and postprandial glucose concentrations, reductions in glycosylated hemoglobin A1C (HbA1c) and reductions in body weight [23,13,55]. Exenatide shares 53% amino acid sequence identity with human GLP-1 [30,18]. In contrast to GLP-1, exenatide with a glycine at position 2 lacks a recognition sequence for the ubiquitous enzyme, dipeptidyl peptidase-IV, increasing its relative resistance to proteolysis. In addition, the glucoselowering activity of exenatide in animal models is up to 3000fold more potent than exogenous GLP-1 [106]. Since exenatide shares sequence similarities with human GLP-1 and glucagon (53% and 45% amino acid identity, respectively), development of autoimmunity and immunoneutralization of these related
peptides 27 (2006) 1902–1910
endogenous peptides by anti-exenatide antibodies are potential concerns. Indeed, loss of GLP-1 activity in baboons using an anti-GLP-1 monoclonal antibody has been shown to reduce postprandial glycemic control and diminish the release of early phase insulin compared to controls [21]. In addition, intravenous infusion of a competitive GLP-1 receptor antagonist in healthy humans resulted in dosedependent antagonism of the insulinotropic and glucagonostatic effects of GLP-1 [94]. Importantly, examination of serum samples from human subjects containing anti-exenatide antibodies revealed no significant cross-reactivity with either glucagon or GLP-1 [44]. Further, the presence of antiexenatide antibodies did not result in worsened glycemia or diminished counterregulation as would be predicted with loss of GLP-1 or glucagon function. Recent reports of placebo-controlled studies of exenatide as an adjunct to metformin [23], sulfonylureas [13] or both [55] indicate that exenatide treatment for 30 weeks led to antibody development in 41–49% of patients irrespective of background therapy (Fig. 1). A majority of patients exhibited low titer (1/ 125) anti-exenatide antibodies at the end of each study, with a minority of subjects (5–7%) displaying antibodies of highertiter (1/625) [13,14,23,55]. In approximately 3% of patients with high-titer anti-exenatide antibodies, an attenuated glycemic response was noted, indicative of some degree of clinical resistance [14]. However, other subjects with higher titer antibodies (>1/625) showed no apparent diminution of glycemic response. Therefore, the presence of anti-exenatide antibodies, even those of high titer, was not indicative or predictive of the magnitude of glycemic effects. Consistent with these findings, 24-week data from an open-label extension study of exenatide on a background of metformin, a sulfonylurea, or both showed that the mean change from baseline in A1C was 1.2% for antibody-negative patients versus 1.4% for patients with detectable antibody titers [77]. Antibody responses were not attributed to a particular immunoglobulin isotype and titers reportedly diminished over time in most antibody-positive patients [14]. Composite data of patients completing 52 weeks of exenatide exposure in open label extensions showed that incidences of antibody-positive subjects increased approximately 4 weeks after treatment initiation, peaking at 4–5 months with approximately 50% of patients antibody-positive, and decreasing to approximately 34% at 1 year (Fig. 1). Findings in this cohort were similar to those observed in the placebocontrolled studies [1]. In addition, levels of eosinophils were measured through the course of the long-term controlled studies as a diagnostic marker of a hypersensitivity response. Circulating eosinophil levels in exenatide-treated patients did not reveal significant leukocyte activation compared to placebo treated patients. These results, along with examination of patient adverse event profiles, were inconsistent with an increased risk of hypersensitivity reactions associated with exenatide treatment [1]. Moreover, the types of adverse events reported in patients with detectable anti-exenatide antibody titers were similar to those observed in antibody-negative patients [14]. To date (2 years of drug exposure), the presence of antiexenatide antibodies has not been associated with adverse clinical consequences.
5.
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Conclusions
Antibody formation is a common response to peptide therapy that potentially may lead to unwanted effects such as decreased drug efficacy or serious adverse events. In a majority of cases, immune responses are benign in nature with minimal clinical impact. Similar to clinical experiences with peptide-based agents in general, a majority of cases of antibody formation in response to animal insulins, animal or fish calcitonins and exenatide have not led to serious clinical sequelae or complications. With the use of highly purified animal (and human) insulins, antibody levels are reportedly lower and allergic reactions such as lipoatrophy are unusual [93]. Currently, immunological complications associated with insulin therapy overall have decreased significantly, including the incidence of insulin-antibody-mediated insulin resistance [33,104]. The most reported outcome of antibody formation during pork or salmon CT therapy is acquired clinical resistance, which has been effectively treated with increased dosage or by switching to human CT. In the case of exenatide, findings to date are not indicative of deleterious effects resulting from the presence of anti-exenatide antibodies but a small proportion of patients have exhibited an attenuated therapeutic response. Long-term studies have demonstrated that the presence of antibodies in response to these xenopeptides persists with continued treatment (Fig. 1). While the differences in sequence identity between xenopeptides and their human counterparts increase the likelihood of a more vigorous immune response, the extent of immunogenicity does not strictly correlate with sequence homology to human proteins. Overall, human therapies tend to be less immunogenic, both in titer and in prevalence, however, they are still immunogenic to some degree. Notably, reported cases of greatest severity and clinical impact resulting from immune reactions to therapeutic proteins have been associated with antibodies against human-derived therapeutics (i.e. rhuEpo). Since drug-induced antibody response and its influencing factors are not fully understood or predictable, investigations of immunogenicity and its clinical impact are an important safety consideration for all forms of protein therapy, particularly those used to treat chronic diseases. Furthermore, drug-induced antibody formation on its own has not provided a strong basis for discontinuing treatment. Rather, demonstration of a clinical correlate in addition to the presence of circulating antibodies is warranted, with drug-induced immune effects defined for each individual therapy.
Acknowledgements We gratefully acknowledge Tom Bicsak for critical reading of the manuscript and Gordon Bedford for library support.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Review
Immunogenicity of xenopeptide hormone therapies Catherine A. Schnabel a,*, S. Edwin Fineberg b, Dennis D. Kim a a b
Amylin Pharmaceuticals, Inc., 9360 Towne Centre Dr., San Diego, CA 92121, USA Indiana University School of Medicine, Indianapolis, IN 46202, USA
article info
abstract
Article history:
Peptides are a growing class of agents whose therapeutic use originated with non-human
Received 2 November 2005
treatments such as animal insulins. Xenopeptides continue to be explored for biothera-
Received in revised form
peutic development using genetic engineering, and through the rich resource of animal and
25 January 2006
plant polypeptides. One of the major concerns of therapeutic administration of xenopep-
Accepted 26 January 2006
tides is the potential for untoward immune responses that may lead to loss of drug efficacy
Published on line 3 March 2006
or adverse events in recipients. An increased risk of immunogenicity is perceived with xenopeptides, however, human-derived therapies also induce antibody formation that in
Keywords:
some cases has been associated with severe clinical sequelae. In this review, antibody
Immunogenicity
responses to xenopeptides are highlighted looking at current hormone therapies used to
Xenopeptide
treat endocrine disorders. Similar to clinical experiences with peptide-based agents in
Hormone therapies
general, antibody responses against xenopeptide hormone therapies in majority of cases have been benign in nature with minimal clinical impact. # 2006 Elsevier Inc. All rights reserved.
Contents 1. 2. 3. 4.
5.
1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immune responses to protein therapeutics . . . Variables of antibody induction and detection . Xenoprotein therapies . . . . . . . . . . . . . . . . . . . . 4.1. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Calcitonin . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Exenatide . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction
Protein therapeutics have revolutionized the treatment of a wide variety of diseases [36]. Because of their specificity,
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1902 1903 1904 1904 1904 1905 1906 1907 1907 1907
peptide-based agents comprise an important component of the therapeutic arsenal, and have the potential to provide efficacy with minimal adverse effects. A significant barrier to the use of protein therapeutics is the development of
* Corresponding author. Tel.: +1 858 458 8464; fax: +1 858 824 7628. E-mail address: [email protected] (C.A. Schnabel). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.01.019
peptides 27 (2006) 1902–1910
unwanted anti-drug immune reactions. Nearly all protein drugs have the potential for antibody induction, although the incidence and associated clinical consequences vary widely among agents [19,45,58,90,91]. In the case of therapies intended for chronic therapy, surveillance for adverse immunological effects are particularly important since they may contribute to complications associated with long-term use. Concern over untoward immunological effects of protein therapeutics was initially brought to the forefront when the first proteins purified from animal tissue extracts became available for therapeutic use [6]. Since non-native molecules were perceived to be more antigenic than their human counterparts, the extent of sequence divergence from human homologs was thought to fully explain their immunogenic properties. However, antibody formation was subsequently shown in recipients treated with protein therapies isolated from human tissues or sera [53,85], including recipients who were not innately deficient for a given protein [93]. Over time, it has become increasingly apparent that factors influencing drug-related immunogenicity are not fully understood, or completely predictable.
2.
Immune responses to protein therapeutics
Immunogenicity of therapeutic proteins has largely been defined by the presence of circulating antibodies in clinical samples. Various assay formats are used to assess immunogenicity. First-line assays used to measure antibody levels include enzyme-linked immunosorbant assays (ELISAs), radioimmunoassays (RIAs) and more sophisticated methods such as surface plasmon resonance (SPR). Additional qualitative information on antibody specificity and immune responses is attainable through lower-throughput methodologies including immunoblotting, ex vivo T-cell activation assays and animal studies in genetically engineered mouse models. Further characterization of potential neutralizing activity is typically achieved through cell-based bioassays. Distinct advantages and disadvantages are associated with the specific assay type, each requiring appropriate design, customization and validation [19,20,59,103]. Immune responses to protein drugs can essentially be viewed as an active immunization or a failure of immune tolerance, depending on whether the immune system encounters a unique presentation of an antigen. B-cell maturation and high affinity antibody production ensue through MHC-mediated antigen presentation, T-cell activation and cytokine stimulation. Therapies of non-human origin, such as those from animals, plants or microbes, elicit the formation of foreign-antigen-specific antibodies. Generally, antibodies mounted against such antigens develop as an IgG isotype upon antibody maturation, and can be produced and persist even after a single dose [91]. In comparison, antibody responses against human proteins result from breaking tolerance mechanisms that exist for self-antigens. Immune tolerance may result from active suppression of Bcell maturation by regulatory T-cells or through the destruction or sequestration of antigen specific T-cells [73]. Disruption of tolerance has been related to alterations in the physical properties of the drug, the spread of immunological sequelae
1903
during intercurrent illness, or the use of immunity-modifying drugs, however, precise underlying mechanisms leading to the breaking of tolerance are not completely understood [8,12,92]. These antibodies may be binding, with or without neutralizing activity, and appear after prolonged treatment [90]. In some cases, anti-drug antibodies are observed in a minority of patients and are undetectable after treatment cessation [99]. The impact of antibody formation against protein therapeutics ranges from having no clinical effect to lifethreatening conditions [58]. Antibody incidences also range widely, with less then 1% observed for some products [43] to >70% for others [42]. Among various protein therapeutics associated with immunogenicity, reports of deleterious effects include those where antibodies neutralize an endogenous counterpart with essential, non-redundant functions. The most notable and severe occurrence of this was an outbreak of antibody-mediated pure red cell aplasia (PRCA) in patients treated with recombinant human erythropoietin (rHuEpo) [4,15]. Between 1999 and 2001, hundreds of cases of erythropoietin-associated red cell aplasia were identified, with a majority of cases involving patients with chronic kidney disease receiving subcutaneous injections of erythropoietin alpha [89]. Anti-rHuEpo antibodies, which neutralized not only the recombinant protein, but also native erythropoietin, led to the absence of red cell precursors in the bone marrow of patients, rendering them transfusion-dependent [15]. A change in formulation in 1998 was thought to be the reason for the induction of PRCA. Reduced incidences of PRCA reported over the last 3 years have been attributed to a change in the route of administration and revised storage and handling protocols [92]. In another example, a small proportion of healthy platelet donors developed thrombocytopenia when treated with Megakaryocyte Growth and Differentiation Factor (PEG-MGDF) [62]. Incidences of drug-induced illness resembling autoimmune disorders have also been described in association with protein therapies [74]. In particular, druginduced lupus has been reported as an adverse effect of antiTumor Necrosis Factor-a agents as well as numerous other non-protein drugs [5,87]. Clinical and laboratory features of drug-induced lupus are similar to systemic lupus erythematosus except that the syndrome typically reverts after druguse is stopped [5,74]. Taken together, these more serious instances, although relatively less common, have raised increasing concern over the potential harmful effects of antibody formation in response to protein therapeutics. Additional complications may arise from IgE-mediated immune responses. Drug-induced allergies vary from resolvable local responses such as skin reactions to systemic reactions such as anaphylaxis that are potentially fatal. Incidences of severe immediate hypersensitivity reactions are relatively rare and more commonly associated with xenopeptides or drug re-exposure [40]. In addition to adverse clinical implications, antibody production may attenuate drug efficacy and alter pharmacokinetic and pharmacodynamic properties. Anti-drug antibodies have been implicated in clinical resistance [42,68,104,101], to the extent that further treatment becomes ineffective even at high doses of the therapeutic agent [7]. Additionally, antigen–antibody complexes are associated with alterations
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in the clearance rate and plasma half-life of drugs, which pose additional safety and efficacy issues [58].
3. Variables of antibody induction and detection Many factors have been implicated in the immunogenic potential of therapeutic proteins (Table 1), which have been reviewed extensively elsewhere [19,58,88,90,91]. Aside from factors that affect the intrinsic response, aspects related to the measurement and characterization of antibody production are also important. Currently, standardization of detection methodologies as well as reporting of antibody levels is lacking [69]. Therefore, comparison of results across laboratories and between manufacturers, and the interpretation of relative immunogenicity, remains a challenge. In sum, drug-induced antibody induction and its associated clinical implications depend on a matrix of factors. A variety of techniques have been developed to address the issue of immunogenicity associated with protein-based therapeutics [19,66]. Aside from humanizing protein sequences in chimeric molecules, efforts to reduce immunogenicity through rational protein engineering have included removal of antibody or MHC-binding epitopes, or sterically inhibiting antibody binding by derivatizing with polyethylene glycol [16,19]. Other patient-based strategies have included prevention of an immune response in recipients by cotreatment with immunosuppressive therapy or by inducing tolerance to limit immune reaction [53]. However, since multiple mechanisms that may or may not be addressable potentially contribute to a drug-induced immune response, long-term monitoring of immunogenicity during clinical trials and during the post-marketing period is essential.
4.
Xenoprotein therapies
The origins of peptide-based therapeutic proteins are as diverse as the many indications for which they have been utilized. Non-human protein sequences from microbes and plants have been used to engineer a variety of hybrid molecules for therapeutic use. The development of chimeric
and humanized monoclonal antibodies has provided unique methods of disease treatment, particularly for antineoplastic and immunological indications. In contrast, protein therapies derived from non-human sources that are essentially homologs or mimetics replicate a native human counterpart in function and regulation, and have been developed as pharmacological replacements. Therapies from non-human sources, or xenoproteins, continue to be explored to exploit novel functions or to provide superior clinical features. A current example of this is desmoteplase, a plasminogen activator (PA) derived from the saliva of the blood-feeding vampire bat (Desmodus rotundus) [3,37]. Desmoteplase exhibits neurotoxicological properties superior to human tissue-PA and is currently being evaluated in clinical trials for the treatment of acute ischemic stroke [41]. The intriguing feature of such agents is that they have been optimized by natural selection, rather than through rational drug design. Antibody responses against hormone therapies have been described [6,35,60,68,86,93,104]. In this review, representative therapies are highlighted to illustrate the implications of antibody formation against xenopeptide hormones currently used to treat chronic diseases.
4.1.
Insulin
Insulin replacement therapy is the mainstay pharmacological treatment for patients with type 1 diabetes and with advanced type 2 diabetes [27]. Immunological complications associated with insulin therapy have been evident since its introduction as a treatment for diabetes in the 1920s. The first insulins were either single species or mixtures of bovine and porcine insulins that were isolated from pancreata. Insulin, which is a 51 amino acid peptide hormone, consists of a 21 amino acid alpha-chain linked by disulfide bonds to a 30-residue betachain. Porcine and human insulin differ by a single amino acid in the beta-chain whereas bovine insulin differs from porcine and human insulin by an additional two amino acids in the alpha-chain [98]. The in vitro use of radioactive insulin in the 1950s showed that virtually 100% of patients treated with animal insulins produced high levels of circulating antiinsulin antibodies [6]. High titer IgG insulin antibodies were shown to bind and neutralize insulin, leading to immunological insulin-resistance by interfering with receptor binding
Table 1 – Factors contributing to increased immunogenic potential of protein therapies Factor
Variable
Increased immunogenic potenial
Intrinsic drug-related factors
Primary sequence Post-translational modification
Non-self antigens Non-native glycosylation
Extrinsic drug-related factors
Manufacturing Purity Formulation Degradation
Presence of contaminants Aggregation Physical, oxidation, deamidation, isomerization
Administration Regimen Method
Longer duration or intermittent treatment Subcutaneous or intradermal > intravenous or oral
Genetic Immune
Specific HLA genotype Impaired or suppressed immunity
Patient-related factors
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[6,101]. Animal insulins were also commonly associated with hypersensitivity reactions of lipoatrophy and urticaria, and IgE-mediated responses were observed in up to 30% of recipients, however, severe systemic immunological reactions were observed in less than 0.1% of patients [22,101]. In addition to amino acid composition, purity was a major problem with early preparations of animal insulins, which were contaminated with several other islet cell peptides such as proinsulin, C-peptide and glucagon [17]. For instance, in a large study of insulin-dependent patients with diabetes, drug-induced autoimmunity was attributed to antibody formation against hormonal contaminants including glucagon, pancreatic polypeptide and vasoactive intestinal peptide in patients treated with conventional versus monocomponent insulin [11]. The prevalence and titers of anti-insulin antibodies have decreased in the last three decades, largely as a consequence of improvements in the purification of insulin preparations and the development of monocomponent insulins [31,101,104]. To a lesser extent, the availability of recombinant human forms of insulin also contributed to decreased immunogenicity. However, numerous studies have confirmed that recombinant human insulin is also immunogenic [31,32,34]. Insulin antibodies were initially detected 1–2 months after starting treatment and were observable at 2 years in some long-term studies [31,32,93,102]. Although animal insulins used for therapy vary by up to three amino acids, studies indicate that the insulin antibodies produced are not specific for variant residues, but react with determinants shared by the human protein [80,49,65]. A study of insulin naı¨ve patients with type 1 or type 2 diabetes treated exclusively with recombinant human insulin reported approximately 40% of patients were antibody-positive at 1 year [32]. In patients with type 1 diabetes, studies have shown a relatively higher incidence of insulin antibodies in children compared with adults after treatment with both human and animal insulins [93,65]. In one study, antibody prevalence rates of approximately 70% and 90% were reported at 1 and 2 years, respectively, in children treated with human semisynthetic insulin [65]. Despite decreased immunogenicity with human insulin, antibody-positive patients continue to be at risk for altered insulin pharmacokinetics and immunological insulin resistance [52]. Clinical consequences of antibody-induced phenomena may include increases in daily insulin requirements, loss of postprandial glucose control and delayed hypoglycemia, but in large scale trials, the presence of anti-insulin antibodies have not been shown to negatively impact long-term glycemic control directly [101]. Both IgG and IgE insulin antibodies have been detected with human insulin treatment, however in low titers and with decreasing incidences in patients treated exclusively with human insulin [102]. Specifically, the high incidence of systemic allergic reactions and immunologicallymediated insulin resistance observed with non-purified bovine or porcine preparations were reported to occur in less than 0.1% of patients treated de novo with human insulin [90,93]. Many studies have compared human insulin with purified porcine insulin [31,32,47,48,65,83]. Comparative studies reveal a general consensus of their similar potency [83,2,46]. In particular, an integrative analysis of results from 45 randomized controlled clinical trials by Richter and Neises com-
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Fig. 1 – Representative profiles of antibody incidences in response to long-term treatment with human (N = 100) or porcine (N = 121) subcutaneous insulin in patients with type 1 or type 2 diabetes, intranasal salmon calcitonin in patients with postmenopausal osteoporosis (N = 44) or exenatide in patients with type 2 diabetes (N = 357). Adapted from Refs. [1,32,82].
pared the efficacy and adverse event profile between human and animal insulins [83]. Most studies did not detect a significant difference in metabolic control, as defined by measures of glycosylated hemoglobin (HbA1c), fasting plasma glucose and insulin use, notwithstanding heterogeneous study designs and demographics [83]. With regard to immunogenicity, studies have demonstrated that insulin antibody levels are lower in patients using human insulin, with approximately 40–50% incidence compared to 60% with purified porcine insulin [31,32,34] (Fig. 1). Interpretations regarding the clinical significance of the decreased immunogenic potential of human insulin have been varied. An early study in children newly diagnosed with type 1 diabetes indicated adverse effects on residual beta-cell function in the presence of low levels of insulin antibodies [64]. In a larger, controlled study, however, differences in beta-cell function could not be correlated to insulin antibody levels comparing newly diagnosed children treated with either human monocomponent insulin or porcine monocomponent insulin [65]. Taken together, comparative studies indicate that the prevalence and titers of antibodies detected in patients treated with human insulin are lower than with porcine insulin, however the decreased immunogenic potential of human insulins generally has minimal effects on metabolic control. In most Westernized countries, the use of animal insulins has been largely replaced with recombinant human insulins.
4.2.
Calcitonin
Effects of calcitonin to suppress osteoclast-mediated bone resorption have led to its use in treating a variety of bone disorders such as Paget’s disease, postmenopausal
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osteoporosis and osteogenesis imperfecta [79]. In patients, long-term treatment with calcitonin has been shown to increase bone mineral density, reduce vertebral and nonvertebral fractures and serve as an analgesic [51]. Calcitonins from several species including human, porcine, salmon and eel have demonstrated clinical value in treating disorders of bone and calcium metabolism [51]. Sequence comparisons among the various therapeutic calcitonins reveal that these 32 amino acid peptides share eight conserved residues (positions 1, 4–7, 9, 28 and 32), with 6 of these clustering at the N-terminus of the molecule [78,71]. Specific divergent residues comprising salmon and eel peptides allow more conformational flexibility, which is a major determinant of biological potency [107]. The activities of salmon and eel calcitonins are approximately 40% more potent than the mammalian calcitonins in lowering plasma calcium and reducing markers of bone turnover [54]. The fish calcitonins, particularly salmon, have achieved more widespread therapeutic use worldwide than human calcitonin due to their increased in vivo potencies and pharmacokinetic properties [50]. Porcine and salmon calcitonins (pCT, sCT) were the first hormones commercially available to treat metabolic diseases characterized by excessive bone resorption [10]. Antibody production during calcitonin therapy was suspected when recipients exhibited a pattern of initial responsiveness with subsequent calcitonin resistance during chronic subcutaneous treatment [95,105]. Studies in patients with Paget’s disease treated with either pCT and/or sCT reported similar prevalences of antibody formation in recipients, ranging from approximately 30% to 40% with sCT and from 60% to 70% with pCT [26,42]. Anti-CT antibodies were detectable approximately 4–12 months after initation of therapy. Upon cessation of therapy, antibodies were undetectable in some patients after 6 months, while in others antibodies persisted for up to 24 months [26]. The most reported outcome of antibody development during heterologous CT therapy is acquired clinical resistance, with antibodies of the IgG class as the major immunological reactants [95,97]. Cases of clinical resistance to sCT are generally correlated to the presence of high-titer anti-sCT antibodies [96]. The clinical significance of CT immunogenicity remains controversial, however, since secondary resistance in some instances is not associated with antibody production [96]. In one study of 15 patients with Paget’s disease treated with pCT or sCT for periods ranging from 4 to 24 months, 6 patients developed secondary resistance related to the presence of neutralizing anti-sCT or anti-pCT antibodies [42]. Another study of 21 patients with Paget’s disease treated with sCT from 1.5 to 19 months reported antibodies in 50% of patients [95]. Whereas three patients developed high titer (>1/ 500) anti-sCT antibodies, only one patient exhibited clinical resistance that could not be explained by dose or treatment duration [95]. Interestingly, cases of secondary resistance to sCT in patients with Paget’s disease have been reported in the absence of circulating anti-sCT antibodies [67], indicating that the occurrence of antibodies does not necessarily correlate with loss of sCT activity. Aside from studies with parenterally administered therapy, the occurrence of antibodies has also been described in
patients treated with nasal sCT [38,61,82]. Reported incidences in osteoporetic patients treated with nasal sCT were comparable to values observed with sCT injection [70]. Similarly, the presence of neutralizing antibodies against sCT administered intranasally in some patients with postmenopausal osteoporosis did not strictly correlate with a loss of therapeutic efficacy [81,82]. Another study of nine patients with Paget’s disease treated for 1 year with nasal sCT showed that all five patients previously treated with injectable sCT developed clinical resistance associated with neutralizing antibody formation within 4 months, indicating that prior exposure may potentiate neutralizing antibody formation [61]. Patients showing clinical resistance to pCT or sCT have been treated effectively with human CT (hCT) [84,63]. Antibody development to hCT is reportedly rare [38,39,97]. Of those reported, antibodies against hCT have been detected in two patients treated for Paget’s disease (one subcutaneous sCT, one intranasal sCT) and in one other treated with subcutaneous sCT for osteoporosis [25,38,39]. sCT therapy currently remains a treatment option for Paget’s disease, osteoporosis and other hypercalcemic conditions. Antibody production against sCT during long-term treatment is undoubtedly common. In general, the presence of low-titer antibodies has no clinical consequences and secondary resistance has been described in the absence of antibodies. Therefore, treatment decisions surrounding sCT should not be dictated solely by the presence of antibodies, but require clinical confirmation evidenced by a parallel loss of therapeutic efficacy.
4.3.
Exenatide
A novel agent recently approved for the treatment of type 2 diabetes, exenatide, is a peptide originally isolated from the salivary secretions of the Gila monster (Heloderma suspectum) [30,72]. Exenatide is a 39 amino acid peptide that shares several glucoregulatory properties with human GLP-1, an incretin hormone released by intestinal cells in response to a meal that augments glucose-dependent insulin secretion [72,75,76]. The actions of exenatide, which target several abnormalities of type 2 diabetes, include the enhancement of glucose-dependent insulin secretion, suppression of inappropriately elevated glucagon secretion, slowing of gastric emptying (which may be paradoxically accelerated in the diabetic state) and reduction in food intake [28,56,57,9,100,29,24]. Improvements in glycemic control with exenatide have been demonstrated as reductions in fasting and postprandial glucose concentrations, reductions in glycosylated hemoglobin A1C (HbA1c) and reductions in body weight [23,13,55]. Exenatide shares 53% amino acid sequence identity with human GLP-1 [30,18]. In contrast to GLP-1, exenatide with a glycine at position 2 lacks a recognition sequence for the ubiquitous enzyme, dipeptidyl peptidase-IV, increasing its relative resistance to proteolysis. In addition, the glucoselowering activity of exenatide in animal models is up to 3000fold more potent than exogenous GLP-1 [106]. Since exenatide shares sequence similarities with human GLP-1 and glucagon (53% and 45% amino acid identity, respectively), development of autoimmunity and immunoneutralization of these related
peptides 27 (2006) 1902–1910
endogenous peptides by anti-exenatide antibodies are potential concerns. Indeed, loss of GLP-1 activity in baboons using an anti-GLP-1 monoclonal antibody has been shown to reduce postprandial glycemic control and diminish the release of early phase insulin compared to controls [21]. In addition, intravenous infusion of a competitive GLP-1 receptor antagonist in healthy humans resulted in dosedependent antagonism of the insulinotropic and glucagonostatic effects of GLP-1 [94]. Importantly, examination of serum samples from human subjects containing anti-exenatide antibodies revealed no significant cross-reactivity with either glucagon or GLP-1 [44]. Further, the presence of antiexenatide antibodies did not result in worsened glycemia or diminished counterregulation as would be predicted with loss of GLP-1 or glucagon function. Recent reports of placebo-controlled studies of exenatide as an adjunct to metformin [23], sulfonylureas [13] or both [55] indicate that exenatide treatment for 30 weeks led to antibody development in 41–49% of patients irrespective of background therapy (Fig. 1). A majority of patients exhibited low titer (1/ 125) anti-exenatide antibodies at the end of each study, with a minority of subjects (5–7%) displaying antibodies of highertiter (1/625) [13,14,23,55]. In approximately 3% of patients with high-titer anti-exenatide antibodies, an attenuated glycemic response was noted, indicative of some degree of clinical resistance [14]. However, other subjects with higher titer antibodies (>1/625) showed no apparent diminution of glycemic response. Therefore, the presence of anti-exenatide antibodies, even those of high titer, was not indicative or predictive of the magnitude of glycemic effects. Consistent with these findings, 24-week data from an open-label extension study of exenatide on a background of metformin, a sulfonylurea, or both showed that the mean change from baseline in A1C was 1.2% for antibody-negative patients versus 1.4% for patients with detectable antibody titers [77]. Antibody responses were not attributed to a particular immunoglobulin isotype and titers reportedly diminished over time in most antibody-positive patients [14]. Composite data of patients completing 52 weeks of exenatide exposure in open label extensions showed that incidences of antibody-positive subjects increased approximately 4 weeks after treatment initiation, peaking at 4–5 months with approximately 50% of patients antibody-positive, and decreasing to approximately 34% at 1 year (Fig. 1). Findings in this cohort were similar to those observed in the placebocontrolled studies [1]. In addition, levels of eosinophils were measured through the course of the long-term controlled studies as a diagnostic marker of a hypersensitivity response. Circulating eosinophil levels in exenatide-treated patients did not reveal significant leukocyte activation compared to placebo treated patients. These results, along with examination of patient adverse event profiles, were inconsistent with an increased risk of hypersensitivity reactions associated with exenatide treatment [1]. Moreover, the types of adverse events reported in patients with detectable anti-exenatide antibody titers were similar to those observed in antibody-negative patients [14]. To date (2 years of drug exposure), the presence of antiexenatide antibodies has not been associated with adverse clinical consequences.
5.
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Conclusions
Antibody formation is a common response to peptide therapy that potentially may lead to unwanted effects such as decreased drug efficacy or serious adverse events. In a majority of cases, immune responses are benign in nature with minimal clinical impact. Similar to clinical experiences with peptide-based agents in general, a majority of cases of antibody formation in response to animal insulins, animal or fish calcitonins and exenatide have not led to serious clinical sequelae or complications. With the use of highly purified animal (and human) insulins, antibody levels are reportedly lower and allergic reactions such as lipoatrophy are unusual [93]. Currently, immunological complications associated with insulin therapy overall have decreased significantly, including the incidence of insulin-antibody-mediated insulin resistance [33,104]. The most reported outcome of antibody formation during pork or salmon CT therapy is acquired clinical resistance, which has been effectively treated with increased dosage or by switching to human CT. In the case of exenatide, findings to date are not indicative of deleterious effects resulting from the presence of anti-exenatide antibodies but a small proportion of patients have exhibited an attenuated therapeutic response. Long-term studies have demonstrated that the presence of antibodies in response to these xenopeptides persists with continued treatment (Fig. 1). While the differences in sequence identity between xenopeptides and their human counterparts increase the likelihood of a more vigorous immune response, the extent of immunogenicity does not strictly correlate with sequence homology to human proteins. Overall, human therapies tend to be less immunogenic, both in titer and in prevalence, however, they are still immunogenic to some degree. Notably, reported cases of greatest severity and clinical impact resulting from immune reactions to therapeutic proteins have been associated with antibodies against human-derived therapeutics (i.e. rhuEpo). Since drug-induced antibody response and its influencing factors are not fully understood or predictable, investigations of immunogenicity and its clinical impact are an important safety consideration for all forms of protein therapy, particularly those used to treat chronic diseases. Furthermore, drug-induced antibody formation on its own has not provided a strong basis for discontinuing treatment. Rather, demonstration of a clinical correlate in addition to the presence of circulating antibodies is warranted, with drug-induced immune effects defined for each individual therapy.
Acknowledgements We gratefully acknowledge Tom Bicsak for critical reading of the manuscript and Gordon Bedford for library support.
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